Proteins are important in biopharmaceuticals as they are widely used to cure a number of diseases including diabetes (e.g. Insulin), cancers (e.g. Interferon, monoclonal antibodies), heart attacks, strokes, cystic fibrosis (e.g. Enzymes, Blood factors), inflammation diseases (e.g. Tumor Necrosis Factors), anemia (e.g. Erythropoietin), hemophilia (e.g. Blood clotting factors), etc. One of the important challenges is the development of efficient and competent process for the large scale purification of these proteins. Numerous processes are available for the large scale purification of the Protein-of-interest from the harvest cell culture fluid (HCCF), but still some impurities remain with the purified Protein-of-interest which can prove to be detrimental to the long term stability as well as quality of the Protein-of-interest. The Protein-of-interest is purified from the HCCF using a series of chromatographic and Ultrafiltration/Dia-filtration techniques.
Although a lot of processes have been developed to purify TNFR:Fc fusion proteins from the HCCF, but due to variability in the cell expression system, it has been observed that general purification processes often fail to adequately purify the Protein-of-interest from the process related impurities. The Protein-of-interest produced by the host cells during cell culture or fermentation has to be purified from host cell-derived proteins (HCP), host-cell DNA, process additives, adventitious agents, toxins and certain product-related substances. These impurities are undesirable in the purified Protein-of-interest and their levels need to be kept within the acceptable levels to render the product safe for human therapeutic use (Wang et. al. 2009 Jun. 15 Biotechnol Bioengineering 103(3):446-58).
Tumour necrosis factor (TNF) is a potent cytokine and elicits a broad spectrum of biologic responses, which are mediated by binding to a cell surface receptor. It is involved in pathogenesis of many inflammatory disorders like rheumatoid arthritis, psoriatic arthritis, SLE, Crohn's disease etc. Hohmann et. al. (Hohmann et. al. 1989 J Biol Chem. 25, 14927-34). Direct inhibition of TNF-alpha by the biological agents has produced significant advances in rheumatoid arthritis treatment and has validated the extra-cellular inhibition of this pro-inflammatory cytokine as an effective therapy. Recombinant TNFR:Fc fusion proteins bind to the cytokine TNF and block the activity of TNF. Examples of TNF-inhibitors include TNFR:Fc fusion protein (Etanercept) and anti-TNF monoclonal antibodies (Adalimumab, Infliximab, Golimumab and Certolizumabpegol).
Etanercept is a dimeric fusion protein consisting of an extra-cellular ligand-binding portion of the human 75 kilo Dalton (p75) tumor necrosis factor receptor (TNFR, type II) linked to the Fc portion of human IgG1. The Fc component of Etanercept consists of the CH2 domain, the CH3 domain and hinge region, whereas the CH1 domain is absent (U.S. Pat. No. 7,648,702). It is produced through recombinant DNA technology in Chinese hamster ovary mammalian cells. It consists of 934 amino acids, and has an apparent molecular weight of approximately 130 kilo Dalton. Due to its unique structure, Etanercept binds more efficiently to TNF alpha than its endogenous receptor (Gofeeet et. al. 2003 J Am Acad Dermatol. 49, S105-111, Strober 2005 Semin Cutan Med Surg. 24; 28-36).
U.S. Pat. No. 7,294,481 discloses purification of TNFR:Fc protein by protein A chromatography followed by hydrophobic interaction chromatography.
EP2729482A1 discloses purification of fusion proteins by protein A chromatography, followed by cation exchange chromatography followed by anion exchange chromatography.
WO2004076485 teaches purification of antibodies by protein A chromatography followed by anion exchange chromatography followed by cation exchange chromatography.
WO2013176754 discloses a method for reducing at least one process-related impurity and/or product-related substance from the Protein-of-interest by hydrophobic interaction chromatography (HIC) in flow through mode.